The increasing demand for therapeutic proteins produced in mammalian expression systems has led in the past 30 years to significant advances in the areas of in vitro cell cultivation, scale-up, expression vectors and medium development.
While ordinary suspension cultures are suitable for growth of certain mammalian cell lines, many cells are anchorage-dependent, and require a solid surface in order to proliferate, metabolize nutrients and produce biomolecules. Conventional means for growth of these cells on a small scale include petri-dishes, flasks and roller bottles. A number of approaches have been taken by various investigators to develop packed bed bioreactors with large surface-area-to-volume ratios for space and operational economy. Another approach is to culture cells on a packing material inside a column shaped vessel that is linked to a bioreactor (Wang et al. 1992).
The use of solid supports such as microcarriers or Fibra-Cel® disks in bioreactor systems increases the available growth area for anchorage-dependent cells per unit of volume and results in obtaining satisfactory protein productivity. Microcarriers and Fibra-Cel® disks are thus routinely used in animal cell culture for the growth of anchorage-dependent cells and are among the established technological platforms for industrial production of proteins (see, e.g., Buck and Loh, J. Viriological Meth., 1985; Bohak et al, Biopolymers, 1987; Petti et al, Biotechnol. Prog., 1994; Ikonomou et al, Biotechnol. Prog, 2002).
Microcarriers are small solid particles on which cells may be grown in suspension culture. Cells are capable of adhering and propagating on the surface of microcarriers. Typically, microcarriers consist of beads, the diameter of which is comprised between 90 μm and 300 μm. Microcarriers can be made of various materials that have proven successful for cell attachment and propagation such as, e.g., glass, polystyrene, polyethylene, dextran, gelatin and cellulose. In addition, the surface of microcarriers may be coated with a material promoting cell attachment and growth such as, e.g., e.g., N,N-diethylaminoethyl, glass, collagen or recombinant proteins. Both macroporous and non-porous microcarriers do exist. Macroporous surfaces give the cells easy access to the interior of the microcarrier after inoculation, and once inside of the microcarrier, the cells are protected from the shear forces generated by mechanical agitation and aeration in the bioreactor.
Fibra-Cel ® disks (New Brunswick Scientific) are disks of 6 mm in diameter that are composed of polyester non-woven fiber bonded to a sheet of polypropylene mesh (see, e.g., U.S. Pat. No. 5,266,476). Fibra-Cel® disks are usually treated electrostatically to facilitate suspension cells adhering to the disks and becoming trapped in the fiber system, where they remain throughout the cultivation process. Cell density and productivity achieved with cells grown on Fibra-Cel ® disks can be up to ten times higher than with cells growing on microcarriers.
Currently, Fibra-Cel® disks and microcarriers are not re-used. However, using new solid supports for each protein production phase is expensive. In addition, changing the solid support after each protein production phase is time-consuming. Accordingly, there is a need for a method of recycling solid supports for cultivation of anchorage-dependent cells in situ, said method allowing maintaining a protein productivity level comparable to the protein productivity level obtained with non-recycled supports.